| The unique features of the brain microvasculature endothelium, also known as the blood-brain barrier (BBB), are imparted by its membrane proteome. The critical role of the BBB membrane proteome in the protection and maintenance of the brain during normal function and in the BBB dysfunction that hallmarks several neurological disorders highlights the importance of BBB membrane proteomic studies. We have developed a novel antibody-based approach, called Multiplex Expression Cloning (MEC), for de novo identification of BBB membrane proteins. MEC involves the mining of a BBB cDNA library, expressed in a mammalian host cell, for membrane protein-encoding clones with the help of a BBB-specific polyclonal antiserum. MEC does not require protein solubilization and yields increased ectopic protein expression levels, thus enabling the detection of even those membrane proteins that due to their hydrophobicity or low abundance levels in vivo are inaccessible to current proteomic approaches such as mass spectrometry. MEC was used to identify a panel of 30 BBB membrane proteins and for the first time, the transcript abundance levels of these membrane proteins were compared across the vascular beds of lung, liver, kidney, heart and brain by use of quantitative polymerase chain reaction (qPCR) coupled with laser capture microdissection (LCM). Five membrane proteins, namely Lutheran, carbonic anhydrase IV, podocalyxin, uncoupling protein 2 and solute carrier family 38 - member 5 were identified as being preferentially expressed at the BBB when compared to other vascular beds and many others displayed unique patterns of vascular expression amongst the tissues tested. Finally, in order to facilitate future profiling of membrane protein expression across tissues or in disease states, a facile platform was developed for the generation of antibody microarrays, by use of yeast surface display. To summarize, this study serves to demonstrate the utility and the power of antibody-based approaches for unraveling the membrane proteome of the BBB, while avoiding some of the pitfalls that confound current proteomic methods. |
